The uncharacterized gene EVE contributes to vesselelement dimensions in PopulusCíntia L. Ribeiroa,b,1,2, Daniel Condeb,1, Kelly M. Balmantb, Christopher Dervinisb, Matthew G. Johnsonc,3,Aaron P. McGrathd, Paul Szewczykd,4, Faride Undae, Christina A. Finegana, Henry W. Schmidtb, Brianna Milesb,5,Derek R. Drosta,6, Evandro Novaesb,7, Carlos A. Gonzalez-Beneckeb,8, Gary F. Petera,b,f, J. Gordon Burleigha,g,Timothy A. Martinb

, Shawn D. Mansfielde, Geoffrey Changd,h, Norman J. Wicketti,j, and Matias Kirsta,b,f,9

aPlant Molecular and Cellular Biology Graduate Program, University of Florida, Gainesville, FL 32611; bSchool of Forest Resources and Conservation,University of Florida, Gainesville, FL 32611; cE. L. Reed Herbarium, Texas Tech University, Lubbock, TX 79409; dSkaggs School of Pharmacyand Pharmaceutical Science, University of California San Diego, La Jolla, CA 92093; eDepartment of Wood Science, Faculty of Forestry, University of BritishColumbia, Vancouver, BC V6T 1Z4, Canada; fGenetics Institute, University of Florida, Gainesville, FL 32611; gDepartment of Biology, University of Florida,Gainesville, FL 32611; hDepartment of Pharmacology, School of Medicine, University of California San Diego, La Jolla, CA 92093; iPlant Scienceand Conservation, Chicago Botanic Garden, Glencoe, IL 60622; and jPlant Biology and Conservation, Northwestern University, Evanston, IL 60208

Edited by Siobhan M. Brady, University of California, Davis, CA, and accepted by Editorial Board Member June B. Nasrallah January 14, 2020 (received forreview July 19, 2019)

The radiation of angiosperms led to the emergence of the vastmajority of today’s plant species and all our major food crops.Their extraordinary diversification occurred in conjunction withthe evolution of a more efficient vascular system for the transportof water, composed of vessel elements. The physical dimensions ofthese water-conducting specialized cells have played a critical rolein angiosperm evolution; they determine resistance to water flow,influence photosynthesis rate, and contribute to plant stature.However, the genetic factors that determine their dimensionsare unclear. Here we show that a previously uncharacterized gene,ENLARGED VESSEL ELEMENT (EVE), contributes to the dimensionsof vessel elements in Populus, impacting hydraulic conductivity.Our data suggest that EVE is localized in the plasma membraneand is involved in potassium uptake of differentiating xylem cellsduring vessel development. In plants, EVE first emerged in strep-tophyte algae, but expanded dramatically among vessel-containingangiosperms. The phylogeny, structure and composition of EVE in-dicates that it may have been involved in an ancient horizontalgene-transfer event.

vessel | xylem | EVE | vessel dimension | phycodnavirus

The development of a vascular system for the efficient trans-port of water and nutrients contributed to the successful

adaptation of vascular plants to a diverse range of terrestrialenvironments. In angiosperms, water transport occurs in highlyspecialized, water-conducting cells in the xylem: The vessel ele-ments. Several vessel elements join vertically to form vessels, themulticellular conduit of water. The physical characteristics ofthese specialized cells have continuously influenced plant growthand evolution, gradually evolving to increase hydraulic efficiency(1). Wider water-transporting tracheary elements of angiospermsprovide lower resistance to water flow and support higher ratesof photosynthesis than those in other living land plants (2, 3).This allowed the development of larger plant bodies, whilemaintaining an optimal balance between hydraulic conductivityand vulnerability to cavitation (4, 5).Genes that contribute to the development of vessel elements

have been identified, including NAC domain transcription fac-tors. The NAC acronym is derived from three genes that wereinitially discovered to contain a particular domain: NAM, ATAF1-2, and CUC2 (6, 7). In vascular plants, genes in the VASCULAR-RELATED NAC-DOMAIN (VND)/NAC secondary wall thick-ening promoting factor/secondary wall-associated NAC domainprotein (SND) clade act as key regulators of xylem differenti-ation (8). The initial differentiation of meristematic cells intovessel elements is followed by cell proliferation, expansion,programmed cell death, and patterned secondary wall deposi-tion (9). It is during this well-coordinated process that the

Significance

Flowering plants exhibit exceptional diversity of form and stat-ure. Their ability to achieve great heights is supported in part bytheir water-conducting specialized cells, the vessel elements,which have gradually evolved and resulted in increased hy-draulic efficiency. Here we report the discovery, functional, andevolutionary analysis of a previously uncharacterized gene,ENLARGED VESSEL ELEMENT (EVE), that contributes to vesselelement dimensions in the perennial woody species Populus.EVE’s appearance among the streptophyte algae and ultimateexpansion in flowering plants may represent an important ad-dition to the genetic toolkit required for plant vascular devel-opment. Surprisingly, EVE homologs are also detected in algae-infecting prasinoviruses, suggesting that it has been horizontallytransferred.

Author contributions: C.L.R., D.C., K.M.B., C.D., M.G.J., A.P.M., P.S., F.U., C.A.F., B.M., D.R.D., E.N.,C.A.G.-B., G.F.P., J.G.B., T.A.M., S.D.M., G.C., N.J.W., and M.K. designed research; C.L.R., D.C.,K.M.B., C.D., M.G.J., A.P.M., P.S., F.U., C.A.F., H.W.S., B.M., D.R.D., E.N., C.A.G.-B., G.F.P., J.G.B.,T.A.M., S.D.M., G.C., and N.J.W. performed research; G.C. contributed new reagents/analytictools; C.L.R., D.C., K.M.B., C.D., M.G.J., A.P.M., P.S., F.U., C.A.F., B.M., D.R.D., E.N., C.A.G.-B.,G.F.P., J.G.B., T.A.M., S.D.M., G.C., N.J.W., and M.K. analyzed data; and C.L.R., D.C., K.M.B.,C.D., M.G.J., A.P.M., P.S., F.U., C.A.F., B.M., S.D.M., G.C., N.J.W., and M.K. wrote the paper.

Competing interest statement: The use of the gene described in the paper to improve plantgrowth and productivity is the subject of a patent (US# 9,650,646) and is currently licensed.

This article is a PNAS Direct Submission. S.M.B. is a guest editor invited by theEditorial Board.

This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Data deposition: Sequences of EVE homologs identified can be accessed from GenBank(https://www.ncbi.nlm.nih.gov/genbank/). Sequences containing the DUF3339 domain areavailable in PFAM (http://pfam.xfam.org/family/PF11820).1C.L.R. and D.C. contributed equally to this work.2Present address: Bayer Crop Science, St. Louis, MO 63167.3Present address: Department of Biological Sciences, Texas Tech University, Lubbock, TX79409.

4Present address: Department of Pathology, University of Cambridge, CB2 1QP Cambridge,United Kingdom.

5Present address: Center for Urban Environmental Research and Education, University ofMaryland Baltimore County, Baltimore, MD 21250.

6Present address: BASF Corporation, West Sacramento, CA 95605.7Present address: Department of Biology, Universidade Federal de Lavras, MG 37200Lavras, Brazil.

8Present address: Department of Forest Engineering, Resources, and Management, Ore-gon State University, Corvallis, OR 97331.

9To whom correspondence may be addressed. Email: mkirst@ufl.edu.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1912434117/-/DCSupplemental.

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dimension of vessel elements is determined. Vessel diameter isuniversally associated positively with stem length in angio-sperms. Thus, the developmental stage that determines vesseldimension is critical to support the size that a given plant canachieve.Potassium (K+) plays a critical active role in plant cell turgor

regulation, including cell expansion during wood developmentin trees, contributing to the size of newly formed vessel ele-ments (10). Moreover, K+ seasonal variation has been observedin poplar cambium, the site of xylogenesis, a high K+ contentoccurs during spring and summer and a significant reductionduring fall and winter (11, 12). Although there is evidence for arole of K+ in wood formation and vessel size in the developingxylem, the cellular mechanism and the transporters relevant toK+ transport and homeostasis, as well as the genetic factorsthat regulate vessel element dimensions, have remained largelyunknown.Here we show that a previously uncharacterized gene,

ENLARGED VESSEL ELEMENT (EVE), contributes to thedimension of vessel elements in poplar trees, impacting hydraulicconductivity, photosynthesis, and growth. Our data suggest that EVEis localized in the plasma membrane and is involved in potassiumuptake in differentiating xylem cells during vessel development. Inplants, EVE appears to have emerged initially in streptophyte algae,and expanded dramatically among vessel-containing angiosperm.The presence of EVE homologs in algae-infecting phycodnavirusessuggests that this gene has also been involved in extensive horizontalmovement in the tree of life.

ResultsEVE Is Involved in the Development of Vessel Elements. To identifygenes that contribute to vessel element dimensions, we measuredvessel diameter in a mapping population of Populus trichocarpa ×Populus deltoids hybrid (13, 14), and identified the most signifi-cant quantitative trait locus (QTL) between genetic markers at

positions 28.5 to 36.9 Mb of chromosome 1 (SI Appendix, Fig.S1). Of the 827 genes within the QTL interval, we previouslyreported 45 (SI Appendix, Fig. S2 and Table S1) as expressedprimarily in wood forming tissues (15), where differentiation ofvessel elements occurs. Variation in xylem mRNA levels of thesegenes was analyzed as a quantitative phenotype (14). Four cis-regulated genes were identified (SI Appendix, Fig. S2 and TableS1), of which Potri.001G329000 was the only one functionallyuncharacterized and, therefore, selected for further analysis.To evaluate the effect of Potri.001G329000 in vessel element

dimensions, we overexpressed it under the control of the 35S pro-moter in hybrid poplar (Populus tremula × alba). We observed thatxylem mean vessel area and vessel count were significantly higher(70% and 35%, respectively) in the three transgenic lines evaluated,compared to wild-type (Fig. 1 A–C and SI Appendix, Figs. S3 andS4). The longitudinal dimension of individual vessel elementsextracted from transgenic lines were also significantly longer (SIAppendix, Fig. S5). We also generated CRISPR/Cas9-mediatedhomozygous mutants (SI Appendix, Fig. S6). Phenotypic resultsfrom three mutant transgenic lines showed the opposite patternrelative to plants that overexpressed Potri.001G329000 (Fig. 1 A–Cand SI Appendix, Fig. S7), with a significant reduction in meanvessel area (18%) and vessel count (31%). Overall, the area occu-pied by vessels was 129% higher in overexpressing and 43% lowerin mutant lines, compared to wild-type trees (Fig. 1D and SI Ap-pendix, Figs. S4 and S7). In contrast to vessel properties, the meanfiber area from transgenic mutant and overexpressing lines was notsignificantly different from wild-type plants (SI Appendix, Fig. S8).Vessel dimension is a plastic phenotype influenced by envi-

ronmental conditions, such as water or nutrient availability (10, 16,17). Thus, changes of molecular or cellular mechanisms that im-pact water or nutrient uptake (e.g., photosynthesis, root systemarchitecture) could potentially impact vessel size indirectly. Toverify that the phenotypes observed in the transgenic plants weredue to the direct effects of the expression of Potri.001G329000

Fig. 1. Vessel number and dimensions are modified in transgenic EVE plants. (A) Xylem cross-section of 2-mo-old, greenhouse-grown P. tremula × alba wild-type and transgenic (CRISPR/Cas9 mutant: eve; overexpression: OX) EVE plants. Cell walls are stained with phloroglucinol and vessels are white. (B) Meanvessel area, (C) vessel count per area, and (D) overall vessel area per unit total xylem area in wild-type and transgenic lines of EVE. Data are presented asmeans ± SE (n = three biological replicates per genotype, with at least three cross-sections measured per replicate). Asterisks indicate significantly lowermeasures in the mutant eve line, and higher in the transgenic OX line, compared to wild-type, at P < 0.001 (one-way ANOVA followed by Dunnett’s test).Results from three independent OX and eve transgenic lines are presented in SI Appendix, Figs. S4 and S6. (E) Correlation between mean individual vesselarea and expression of EVE in a genetically unstructured population of P. trichocarpa. Area of individual vessel elements were measured in partial stemsections of 96 unrelated P. trichocarpa trees grown in common garden for 4 y. Transcript abundance of EVE was quantified in each individual tree by RNA-sequencing, and the normalized expression was determined by the number of fragments per kilobase of transcript, per million mapped reads. Linear modelsfitting vessel phenotypes (number and area) and PotriEVE transcript abundance were generated in Prism 6.01 software. The slope of this model was sig-nificant nonzero (P < 0.0001). ***P < 0.001 (one-way ANOVA followed by Dunnett’s test).

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within vessel-forming cells, we quantified vessel element diameterin in vitro root culture. Roots were generated by Agrobacteriumrhizogenes transformation using leaf explants of wild-type, over-expressing, and mutant plants. After 4 wk of growth in the dark,we observed similar phenotypes in hairy roots as those observed inwhole plants. A significant increase (39%) and decrease (23%) invessel diameter was detected in the overexpressing and mutantlines, respectively, compared to wild-type (SI Appendix, Fig. S9).To further verify the role of Potri.001G329000 in vessel devel-

opment, we also examined the correlation of its expression withindividual vessel area variation in a natural, genetically unstruc-tured population of P. trichocarpa (18). A highly significant, positivecorrelation (r2 = 0.27, P < 0.0001), confirmed that the expression ofPotri.001G329000 impacts vessel dimension (Fig. 1E).These molecular and quantitative genetic approaches led us to

conclude that Potri.001G329000 contributes to the differentia-tion and expansion of cambial derivatives into vessel elements inPopulus, affecting the number and dimension of these cells.Consequently, we renamed it EVE.

EVE Is a Membrane Protein That Forms a Pentameric Complex. TheP. trichocarpa EVE (PotriEVE) coding sequence translates into a70-amino acid protein, consisting almost entirely of the 68 residuesof the DUF3339 domain (Fig. 2A), with a theoretical mass of 9.295kDa. PotriEVE is predicted to contain two transmembrane do-mains of 20 and 23 residues but no cleavable signal peptide (Fig.2A and SI Appendix, Fig. S10). To confirm that EVE is localized inthe membrane, we overexpressed the EVE homolog of Physcomi-trella patens (PpatEVE) (Fig. 2B) in Pichia pastoris in a heterolo-gous expression system. Western blot analysis showed thatdetergent solubilization of membrane proteins from the disruptedPichia lysate is required for visualization of EVE, indicating itspresence in the Pichia membrane (Fig. 2B). For further proteincharacterization, PpatEVE overexpressed in Pichia was purified toyield highly pure and monodispersed protein. PpatEVE proteinwas then analyzed using size-exclusion chromatography in combi-nation with multiangle laser light scattering (SEC-MALLS) (Fig.2C). This allowed us to determine the molecular masses of theEVE protein complex (4.708 ± 0.1 kDa). Overexpression ofPpatEVE in Pichia and exclusion chromatography suggest that EVEexists as a membrane conjugate likely consisting of five molecules.To verify the subcellular localization of EVE in planta, we carried

out a Western blot analysis of soluble and membrane fractions fromtobacco plants transiently expressing the EVE:FLAG fusion pro-tein, which indicated the presence of EVE in the membrane frac-tion (SI Appendix, Fig. S11A). To resolve the membrane location ofEVE, we performed a sucrose density-gradient fractionation, andidentified fractions associated with each specific membrane usingknown markers. The fractions containing EVE overlapped largelywith those associated with the plasma membrane, suggesting lo-calization in that compartment (SI Appendix, Fig. S11B). Finally, weanalyzed tobacco epidermis cells transiently expressing the fusion ofEVE and tdTOMATO fluorescence protein (EVE:tdTOMATO:EVE) and detected similar subcellular localization as the full-length fusion of the aquaporin PIP2A to mCherry, which occurs inthe plasma membrane (SI Appendix, Fig. S11 C–F).

EVE Is Expressed Primarily in Differentiating Xylem and Is Activatedby SND1. By qRT-PCR analyses we observed that, among allhomologs identified in the P. trichocarpa genome, EVE is themost highly expressed in differentiating xylem (SI Appendix, Fig.S12). EVE is also the most consistently, highly expressed gene in thisfamily, in the cell layers that extend until the region of vessel cellexpansion (19). EVE protein localization was verified by immuno-localization, showing its presence in the vascular cambium and initiallayer of dividing and expanding cells of xylem, where vessel forma-tion occurs (Fig. 2D). Since the Populus antibody also recognizes theepitope in the soybean (Glycine max) homolog of EVE, it was

also used for immunolocalization in this herbaceous specieswhere it was present in xylem cells differentiating into vessels(Fig. 2E). High-magnification images show the presence ofEVE in fibers and developing vessels, before they are fullyformed (SI Appendix, Fig. S13).In Arabidopsis and Populus, the differentiation of the vascular

cambium into xylem is regulated by secondary wall-associatedNAC domain transcription factors (8, 20–22). Transcriptomicsstudies in Arabidopsis demonstrated that EVE homologs are directtargets of the SND1 (20), a key transcriptional switch that activatesdifferentiation of xylem cells. We evaluated if PotriEVE is regulatedby this and other secondary wall-associated NAC domain tran-scription factors by transactivation assays in vivo. We usedNicotianabenthamiana leaves to evaluate the transcriptional induction ofthe LUCIFERASE enzyme, driven by the PotriEVE promoter, inthe presence and the absence of Populus SND1 homologs, and twoother transcriptional regulators involved in vessel development(VND6 and VND7). Of all of the transcriptional regulators eval-uated, only SND1 showed significantly higher activation of theLUCIFERASE, compared to the control (Fig. 2F). While inArabidopsis these transcription factors exhibit fiber- (SND1) or vessel-(VND6, VND7) specific expression, in the woody P. trichocarpaSND1 orthologs (PtrWND1A&B, PtrWND2A&B) are expressed inthe vessels, xylary fibers, and ray parenchyma cells in developingxylem (23). Expectedly, we found that the PotriEVE promotercontains a palindromic 19-bp consensus sequence secondary wallNAC binding element to which SND1 binds (24).

EVE Contributes to K+ Uptake. Potassium (K)+ concentration isdistinctly higher in vessels compared to fibers (10). Furthermore,K+ contributes to vessel expansion by regulating it osmotically(11). Because high concentration of K+ occurs where EVE islocalized (cambial and differentiating xylem zones), and EVE’simpact on vessel dimensions, we hypothesized that it is involvedin K+ uptake. To evaluate this hypothesis, a complementationassay was performed using the Escherichia coli strain LB2003, aK+-uptake–deficient triple mutant. Cells grown in low K+ mediashowed growth complementation by expression of EVE (Fig.2G) that parallels the growth observed with the Arabidopsis two-pore potassium channel AtTPK1 (25), used as a positive control.To further verify that EVE is implicated in K+ uptake, we quan-

tified the levels of potassium in vessels of EVE-overexpressing lines,CRISPR/Cas9 mutants and wild-type plants grown in hydroponicliquid nutrient solution. Initial levels of potassium (3.75 mM) weredecreased to 0.375 mM after 2 wk. Three weeks later, potassium wasmeasured using energy-dispersive X-ray spectroscopy (EDS). X-raycounts of K+ were significantly higher in the overexpressing trans-genic lines, compared to the CRISPR/Cas9 mutants and wild-typeplants (Fig. 2 H and I), suggesting that EVE contributes toK+ uptake. While EVE appears to be involved in the uptake of K+,its specific functional role in that process remains unknown.

Plants Overexpressing EVE Have Higher Height Growth and NetPhotosynthesis. Wider vessels are expected to provide lower re-sistance to water flow in the plant vascular system. To evaluatethis hypothesis, we measured hydraulic conductivity in wild-typeand transgenic poplar overexpressing PotriEVE and verified asignificant increase in hydraulic conductivity (∼28%) (Fig. 3A).Wider vessels also support increased transpiration rates associ-ated with higher stomatal conductance and photosynthetic rates,and are highly positively correlated with stem length (height) inangiosperms (26). Thus, we evaluated whether larger vessel areacontributed to stem height growth and net photosynthesis inthree overexpressing and wild-type lines, under conditions ofhigh evaporative demand (diurnal temperature range of 29 to35 °C). Height growth and net photosynthesis were significantlyhigher in plants overexpressing EVE compared to wild-type (Fig.3 B and C). This was not due to differences in biochemical

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Fig. 2. EVE predicted protein structure, cellular localization, regulation, and activity. (A) The PotriEVE is composed almost entirely of the domain DUF3339with two predicted transmembrane domains. (B) Western blot of P. patens EVE in P. pastoris cells. Detergents are needed to solubilize the membrane prior the visu-alization of EVE. (C) Chromatographs from SEC-MALLS analysis of PpatEVE show UV (blue), refractive index (RI, gray), and light scattering (LS, green) detector readingsnormalized to the peak maxima (left axis). The thick lines indicate the calculated molecular masses (right axis; kDa) of the complete protein/detergent complex (red,681.8 ± 15.9 kDa), as well as the contributions of the detergent (green, 623.7 ± 22.2 kDa) and EVE protein complex (blue, 4.708 ± 0.1 kDa) throughout the elution peaks.The theoretical molecular mass of the PpatEVE monomer is 9.295 kDa, suggesting that the EVE protein complex is composed of five units (47.08/9.295 ∼ 5). (D) EVEimmunolocalizations in 2-mo-old plants of P. tremula × alba stem cross-sections show the protein in the differentiating xylem in wild-type plants, while the signal isabsent in eve trees. (E) EVE localization in 1-mo-old plants of G. max cross-sections also reveals EVE in the differentiating xylem. (F) Effect of PotriSND1, PotriVND6 andPotriVND7 on the regulation of PotriEVE. 35S::GUS::tNOS and pEVE::LUC::tNOS were used as controls. LUC and GUS enzymatic activity was expressed as LUC/GUS relativefluorescence units in each biological replicate, normalized to the wild-type control. Data are presented as means ± SE (n = five biological replicates for each effector/reporter combination). Statistical differences between effector and control were determined by one-way ANOVA followed by Dunnett’s test. **P ≤ 0.01. (G) A com-plementation assay using the E. coli strain LB2003 showed growth complementation in cells growing in low K+media (3 mMK+) by expression of EVE and AtTPK19. Theplotted values are the means ± SD of three biological replicates. (H) Carbon-coated stem cross-sections of EVEOX, CAS9, and wild-type lines were examined in a TESCANMira 3 electron microscope fitted with an EDAX Octane Pro X-ray analyzer. For each vessel, the characteristic peak of the X-ray counts of potassium was recorded. In thegraph, data from an individual vessel for OX, wild-type, and Cas9 lines are plotted. (I) The average of the counts peak for potassium recorded in three biological replicatesof OX, wild-type, and Cas9 lines showed significantly higher potassium content in EVE OX lines, compared to wild-type trees (one-way ANOVA followed by Dunnett’stest). Data are presented as means ± SE. ***P ≤0.001. Results from two independent OX and Cas9 transgenic lines are presented in SI Appendix, Fig. S14.

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photosynthetic capacity between the lines, as measured by plotsof net CO2 assimilation rate versus calculated substomatal CO2concentration, but strictly to stomatal closure limitations in thewild-type trees (SI Appendix, Table S2). In contrast, lowerevaporative demand conditions (diurnal temperature of 25 °C)resulted in no significant phenotypic differences in photosyn-thesis or growth between transgenic and wild-type plants.

EVE Appeared Prior to the Colonization of Land and May HaveOriginated From a Horizontal Gene Transfer. The development ofan improved vascular system had a significant role in the evolutionof plants. Thus, we evaluated the occurrence of EVE-like genesacross plant evolution and detected putative homologs in nearly allland plant genomes sequenced to date (SI Appendix, Table S3), withnotable absences in the ferns Azolla and Salvinia. We supplementedour database with transcriptomes from hornworts and green algae(OneKP.org) (27). This allowed us to identify putative homologs inall three bryophyte lineages (one to three copies) and all seedplants. Compared to other land plants, a large expansion and di-versification of EVE-like genes occurred among most floweringplants (monocots have 5 to 19 copies, eudicots 2 to 20 copies).To investigate the origin of EVE, we expanded our search for

possible ancestors in taxa that precedes the land plants evolu-tionarily. We detected several EVE-like genes in streptophytealgae (28) (SI Appendix, Table S3). Streptophyte algae are a

paraphyletic grade of algae, one lineage of which represents theclosest relatives to land plants. This observation points to theappearance of EVE among green plants prior to colonization ofland. The search in chlorophyte genomes, a clade of green algaedistantly related to land plants, as well as Bacterial or Archaeadomains, showed no evidence of EVE. Surprisingly, EVE-likegenes were identified in numerous prasinoviruses of the Phy-codnaviridae family and in one species of amoebae (Acantha-moeba castellanii). Prasinoviruses, also known as “giant viruses,”are lytic and lysogenic viruses with a genome ranging from 160 to560 kb (29).To further evaluate homology, we searched and confirmed

that EVE-like genes in Phycodnaviridae contain the characteristicfeatures of the DUF3339 domain present in land plants (http://pfam.xfam.org/family/PF11820). A phylogeny of EVE-like genesacross viruses, algae, and land plants was constructed. The treerevealed a close phylogenetic relationship between viral andstreptophyte copies (SI Appendix, Figs. S15 and S16).The absence of putative EVE homologs in chlorophyte algae,

together with the closer phylogenetic relation between viral andstreptophyte algae, is suggestive of a horizontal gene transfer(HGT) event between prasinoviruses and an ancestral streptophyte.The length of putative EVE homologs is significantly shorter thanthe average gene size in plants, but similar to that of prasinoviruses(Fig. 4A). To further evaluate the HGT origin of EVE, we in-vestigated nucleotide base composition in the genome of Klebsor-midium nitens (28), which shares the most ancient common ancestorof EVE putative homologs in plants (Fig. 4B). We identified a highlysignificant genomic islands (discrete interval accumulative score[DIAS] = 12) possibly originated by HGT, containing the algaeEVE homolog (Fig. 4 B and C). To discard the possibility that thepresence of an EVE-like gene in the K. nitens genome is due tocontamination with prasinoviruses, we confirmed the presence ofthe EVE genomic island within the K. nitens genome by PCR inindependent algae cultures (SI Appendix, Fig. S17).

DiscussionWe performed QTL and xylem transcriptome analysis of amapping population of P. trichocarpa × P. deltoides and identifieda previously uncharacterized gene, EVE, involved in vessel devel-opment. Analysis of vessel dimensions in transgenic poplar treesoverexpressing EVE and CRISPR/Cas9-mediated homozygous mu-tants, as well as transcript variation of EVE in a natural populationof P. trichocarpa, showed significant changes of vessel area that arecompatible with a role in the regulation of vessel dimensions. In-terestingly, CRISPR/Cas9-mediated homozygous mutants didn’tabolish entirely the occurrence of structurally normal vessel ele-ments. This may have occurred because of functional redundancyfrom EVE-like genes expressed in differentiating xylem, or thecompensatory contribution from other genes with a similar function.The observation that EVE complements the growth pheno-

type of the E. coli strain LB2003, a K+-uptake–deficient triplemutant, suggests a functional role in cell K+ uptake. K+ is the mostabundant cation in higher plants, and is the major inorganic, os-motically active substance in differentiating cambium cells (30,31). K+ has a critical role in numerous aspects of xylogenesis, suchas cell expansion through an increase of symplastic K+ content andxylem development (11). In plants growing under nonlimitingK+ supply, the developing xylem zone and the cambial K+ levelsare significantly higher than in plants growing under limiting K+

(10). An increase in K+ supply also results in an increase of vesselsize; and treatment with tetraethylammonium, a K+ channelblocker, significantly reduces the size of newly formed vessels (10).To further verify the role of EVE in K+ uptake, we performed anEDS assay, where plants were grown under low K+ conditions(0.375 mM KCl) (12, 32). Under these conditions, overexpressinglines showed a significant increase in K+ concentration in thevessels, compared to wild-type plants. In contrast, no difference

Fig. 3. Transgenic plants overexpressing EVE have higher hydraulic con-ductivity and support higher growth and photosynthesis under high evap-orative demand. (A) Maximum hydraulic conductivity (means ± SE) issignificantly higher in overexpressing transgenic EVE, compared to wild-typetrees, when grown at 29 to 35 °C. (B) Net photosynthesis rate (means ± SE) issignificantly higher in all overexpressing transgenic EVE lines compared towild-type trees at 10:00 AM. At 2:00 PM, 6:00 PM, and 10:00 PM, net pho-tosynthesis rate is significantly higher in one or more overexpressing trans-genic lines compared to wild-type. Net photosynthesis rate was measuredrepeatedly on the same plants during the experiment, on six trees per ge-notype. For (A and B), asterisks indicate statistically significant differencebetween each transgenic line and wild-type, at **P < 0.01 and *P < 0.05 bytwo-tailed Student’s t test. (C) Overexpressing transgenic EVE lines maintainactive growth and higher height growth rates of each transgenic line com-pared to wild-type. The plotted values are the means ± SE. The gray boxindicates the growth phase where the overexpressing transgenic lines out-performed wild-type trees and showed a significantly higher growth rate atP = 0.05 (two-tailed Student’s t-test). Measurements were taken repeatedlyon the same plants during the experiment, on six trees per genotype. (D)Wild-type and overexpressing transgenic EVE lines final growth after 14 wkin growth chamber.

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was detected between CRISPR/Cas9 mutants and wild-type. Thissuggests that, under these growth conditions, the absence of afunctional copy of EVE may be compensated by other potassiumtransporters or EVE-like family members. The functional role ofEVE under different levels of K+ availability, and the implicationsto vessel dimensions, should be evaluated in the future.Results of the localization experiments showed that EVE is

not expressed exclusively in cambium cells differentiating intovessels. Instead, expression was also detected in fiber cells of thedifferentiating xylem. However, we did not observe any differ-ence in fiber structure in the transgenic plants, compared to wild-type. The absence of differences between fibers of transgenic andwild-type plants suggests that EVE may promote K+ uptake inboth developing fibers and vessels, but the presence of an out-ward K+ channel expressed only in fibers could compensate forthis uptake. PTORK, which is absent in vessels but expressed innew fibers, could play such a role by mediating K+ efflux (33). Inthis scenario, potassium released from fibers could further con-tribute to its accumulation in differentiating vessels.To further uncover the cellular role of EVE, we performed

heterologous overexpression in yeast and observed its localizationin the membrane. Exclusion chromatography suggests that it existsas a conjugate of five copies. Experiments designed to evaluate thelocalization of EVE in planta pointed to its presence in the plasmamembrane. Finally, we showed that EVE expression is directlyactivated by the poplar SND1 homolog, which is a regulator ofvascular development. The combination of structural, spatial, andfunctional data suggests that EVE influences vessel dimensions byphysical uptake of K+ during the process of cell expansion in thedifferentiating xylem.Our measurement of water conductivity showed, as expected,

that the larger vessels of EVE-overexpressing lines lead to moreefficient water transport. Interestingly, we observed that underhigh temperature, plants overexpressing EVE exhibited signifi-cantly greater height growth and net photosynthesis. Thus, thehigh evaporative demand triggered by warmer temperatures wascompensated by the improved hydraulic conductivity in plantsoverexpressing EVE, possibly by allowing stomata to remain openunder increased evaporative demand. These results are consistentwith the previously reported linear relationship between stem hy-draulic conductivity and growth (34–42). Our results indicate that,in the absence of water deficit-mediated cavitation, wider vesselslead to higher growth rates under high evaporative conditions, inagreement with the hypothesis that vessel widening contributed tothe expansion of land plants with larger body size (4, 5).

In the search for the evolutionary origin of EVE, we showedthat putative homologs are present in most land species and inthe streptophyte algae, which are the closest aquatic relatives toland plants. This result points that the origin of EVE precedes landcolonization by plants. However, the functional role of EVE inalgae and land plant species that lack a vascular system remainsunknown. An evaluation of the number of copies showed that alarge expansion and diversification of EVE-like genes occurredamong most flowering plants, which coincides with the expansionof the vessels across land plants (43). Thus, the functional roleof EVE in vessel development is derived from its function instreptophyte algae and nonvascular land plants. The increasednumber of EVE-like genes in conifers suggests that the expansionis coincident in evolution with the establishment of the secondarygrowth mediated by the vascular cambium in land plants (43). Inthe future, the functional role of EVE in the water-conductingcells of conifers, the tracheids, should be evaluated.Surprisingly, the search for EVE putative homologs resulted in

its detection in Prasinovirus, a genus of the Phycodnaviridae family,which infects eukaryotic algae from both fresh and marine waters,and often integrates part of their genome into that of their hosts(44, 45). Phycodnaviruses belong to the monophyletic clade ofnucleocytoplasmic large DNA viruses (NCLDVs), characterizedby a large double-stranded DNA genome (46, 47). Recent studiesshowed that certain eukaryotes contain fragments of NCLDVDNA integrated to their genome, when surprisingly many of theseorganisms were not previously shown to be infected by NCLDVs(48). The eukaryotic groups most impacted with the presence ofNCLDV viral homolog genes are brown algae (Phaeophyceae),Amoebozoa, and green algae (Chlorophyta and Streptophyta). Of 11genomes available for amoebas (amoebadb.org), one contains aDUF3339 copy, A. castellanii. In fact, the A. castallanii genomesequencing revealed multiple fragments of prasinoviruses DNAintegrated in its genome (49). The Acanthamoeba genus is one ofthe most efficient laboratory hosts to isolate giant viruses fromaquatic samples (50–53). Taking these data together, we show thatthe presence of EVE copies in green algae and amoeba suggeststhat multiple HGT events occurred between an ancestor of theprasinovirus lineage and an ancestral streptophyte and amoeba,likely facilitated by sharing the same aquatic habitats.

Materials and MethodsAdditional information can be found in SI Appendix, Material and Methods.

QTL Analysis of Vessel Development. A hybrid Populus backcross populationof 397 individuals was genotyped as described previously (54). Cross-sectionswere obtained from the stem harvested from 100 individuals representing

Fig. 4. HGT origin of EVE. (A) Box-plot describing the length of all genes in Phycodnaviruses (Phy), in the earliest plant species known to contain a putativeEVE homolog (K. nitens, Kn), and the length of EVE-like genes across plant species (EVE), showing that EVE structure in plants resemble other viral genes. (B)SigHunt analysis of the K. nitens genome identifies a highly significant genomic island associated with a HGT event, at the location of the putative EVEhomolog. The 1-kb window containing EVE was scored based on the number of the 4-mer frequencies that deviate from the credibility interval of their localgenomic density, using a DIAS. (C) Density of DIAS in the K. nitens genome shows that high scores, such as that observed in the location of the putative EVEhomolog, are rare.

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the most informative recombinations. Vessels were measured by visualizingthe sections in a microscope and analyzing the images obtained. Individualvessel areas were converted to diameters (d) and counted (n), and meanhydraulic diameter (Dh, μm, [(Σd4)n-1]1/4) was calculated (2). QTL analysis ofvessel mean hydraulic diameter (Dh) was carried out using composite intervalmapping implemented in QTL Cartographer v2.5 (55).

Generation and Phenotyping of Transgenic Plants Overexpressing EVE andCRISPR/Cas9 EVE Mutant Plants. Overexpressing transgenic lines were gener-ated with EVE under control of the 35S cauliflower promoter. For generation ofCRISPR/Cas9 EVE mutant plants, a guide RNA (gRNA) targeting EVE was selectedfrom http://aspendb.uga.edu/ (56), and cloned as previously described (57). Con-structs were transferred into Agrobacterium and transformation of the hybrid(P. tremula ×alba) poplar genotype 717-1B4was performed (58). Vessel propertieswere measured as described above. Measurement of vessel element longitudinaldimensions and stem fibers was achieved by isolating them (59), visualizing themin a microscope and quantifying their properties. Vessel elements of transgenicpoplar hairy roots were characterized using a similar approach.

Measurement of Hydraulic Conductivity. Conductivity was measured in stemsegments connected to a low-pressure flow apparatus (60). Flow of a perfusionsolution through the stems was performed at low pressure (4 kPa), and weightof the receiving container was recorded. Based on these data, the initial“native” conductivity (Knative) was calculated according to Darcy’s law (2). Afterremoval of all embolisms in the vascular system, the flow was recorded again andconverted to maximum conductivity (Ks max, kg s−1 MPa−1 m−1).

Measurement of Vessel Properties and EVE Expression in a P. trichocarpa AssociationPopulation.Vessel phenotypes and PotriEVE gene expressionweremeasured in 96unrelated, 4-y-old black cottonwood (P. trichocarpa) genotypes grown in acommon garden, as described previously (18). Cross-sections were obtained andvessel properties were quantified as described above. RNA from xylem scrapingscollected from the stem of each tree was purified and quantified prior to RNA-sequencing (RNA-seq) library preparation and sequencing in an Illumina HiSeq.2000. The raw RNA-seq data were analyzed as described previously (61).

Measurement of Growth and Photosynthesis under Normal and High EvaporativeDemand in Transgenic Poplar Plants. Two temperature experiments were con-ducted in a growth chamber, with plants grown under soil moisture saturationand long day lengths. Following acclimation at 25 °C for 6 wk, plants weregrown either under normal conditions (20 °C to 25 °C) or under high evapo-rative demand (29 °C to 35 °C). The diurnal net photosynthesis rate and curvesof net photosynthesis relative to leaf internal CO2 concentration (A-Ci curves)were measured on the first fully expanded leaves. The maximum rate of car-boxylation of Rubisco (Vcmax) and maximum rate of electron transport (Jmax)were derived from A-Ci curves, as described previously (62).

EVE Purification and Structure Analysis. The EVE gene from P. patens (PpatEVE)was cloned into the pPICZc vector and expressed using P. pastoris prior topurification by nickel affinity chromatography and SEC. The structure ofPpatEVE was analyzed using SEC-MALLS.

RT‐PCR Expression Analysis of EVE and Its Homologs in Differentiating Xylem ofP. trichocarpa. Differentiating xylem was collected from Populus wild-typeplants and RNA was extracted (63). qRT-PCR analyses were performed andgene expression was quantified as described previously (64).

EVE Antibody Synthesis and Immunohistochemical Analysis. EVE antibodieswere made by Genscript. For immunohistochemical analysis, basal segmentsof stems of Populus wild-type, EVE homozygous CRISPR/Cas9 mutant linesand soybean (G. max) wild-type were formaldehyde-fixed and preserved, aspreviously described (65). Segments were then sectioned and permeabilizedbefore incubation with anti-EVE antibody and the corresponding secondaryantibody, prior to washes and visualization under a confocal microscope.

Subcellular Localization of EVE. Golden Gate technology was used to assemblep35S::EVE:FLAG::t35S transcriptional unit and clone it into the destinationvector pAGM4673 (66). A set of binary vectors containing organelle-targeting proteins fused with mCherry were acquired from the ArabidopsisBiological Resource Center (The Ohio State University, Columbus, OH). To verifythe subcellular localization of EVE, we expressed transiently the constructsdescribed above in N. benthamiana leaves through agroinfiltration, and per-formed a sucrose density-gradient fractionation, as previously described (67).Subcellular localization of EVEwas also determined usingmicroscopy. The GoldenGate system was employed to assemble the p35S::EVE:tdTOMATO:EVE::t35Stranscriptional unit. After agroinfiltration, leaves were sectioned and placedunder the confocal microscope for visualization of the fluorescence emittedby tdTOMATO and mCherry.

Growth ComplementationAssay.Competent LB2003 cellswere transformedwith aplasmid carrying the coding sequence of EVE, a plasmid carrying the codingsequence of anArabidopsis two-pore potassium channel (AtTPK1), and an emptyvector. Cells were grown overnight, harvested, and washed to remove any re-sidual potassium. Expression of EVE and AtTPK1 was induced by isfopropyl-β-D-thiogalactopyranoside (IPTG) and OD600 values were obtained for each genotype(EVE, AtTPK1, and empty vector) every 2 h for a period of 32 h.

EDS Assay. Plants from EVE CRISPR/Cas9 mutant lines, overexpressing trans-genic lines and wild-type were grown in aerated Hocking’s Complete Solu-tion (Hocking, 1971) containing 3.75 mM KCl for 2 wk. Next, plants weretransferred to a solution containing 0.375 mM of KCl. After 3 wk, internodecross sections were obtained, freeze-dried, coated with carbon, and exam-ined in a TESCAN Mira 3 scanning electron microscope fitted with a EDAXOctane Pro X‐ray analyzer. The potassium content of vessels was measuredas the potassium counts peak (minus background) recorded after 50 s.

Transactivation Assay. PtaSND1 (Potri.011G153300), PtaVND6 (Potri.012G126500),and PtaVND7 (Potri.013G113100) were amplified from P. tremula × alba cDNAsamples and a 2,000-bp fragment of the PtaEVE promoter was amplified fromP. tremula × alba gDNA. Fragments were purified and cloned into the pUPD vector.Golden Braid technology (68) was employed to place the 35S::PtaSND1, PtaVND6or PtaVND7::tNOS (effector) together with 35S::GUS::tNOS (reporter) andpEVE::LUC::tNOS in the 2omega1 plasmid. Transient N. benthamiana agro-infiltration experiments were completed as previously described (69). N. ben-thamiana leaves were collected 3 d postinfiltration for protein extraction andGUS or LUC enzymatic activity measurements.

Evolution of EVE. We searched for EVE in previously sequenced plant ge-nomes and transcriptome data from the One Thousand Plant TranscriptomeProject (1KP, onekp.com). From the final set of detected sequences withsimilarity to EVE, copy number in each lineage was tabulated. Maximum-likelihood analysis of 56 plant DUF3339 proteins from representative specieswas conducted with RAxML v. 8.2.12 (70).

Identification of Genomic Islands due to HGT. SigHunt (71) was employed toidentify genomic islands originated from HGT events in the K. nitens genome(28). We estimated 4-mer density and its variance in sliding windows of chro-mosome DNA sequence, as described previously (71). The windows were thenscored using a DIAS with the improved variant of the method (71).

Data Availability. Sequences of EVE homologs identified can be accessed fromGenBank (https://www.ncbi.nlm.nih.gov/genbank/). Sequences containing theDUF3339 domain are available in PFAM (http://pfam.xfam.org/family/PF11820).

ACKNOWLEDGMENTS. We thank Pieter Bass, Ron Sederoff, Pamela Soltis,and Douglas Soltis for critical reading of the manuscript; and Thomas Woodfor the gift of the LB2003 Escherichia coli. This work was supported by the USDepartment of Agriculture Plant Feedstock Genomics for Bioenergy Program(Grant 2009-65504-05697) and the Department of Energy Office of ScienceBiological and Environmental Research (Grant DE-SC0003893).

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